H2O2 selectively damages the binuclear iron-sulfur cluster N1b of respiratory complex I

NADH:ubiquinone oxidoreductase, respiratory complex I, plays a major role in cellular energy metabolism by coupling electron transfer with proton translocation. Electron transfer is catalyzed by a flavin mononucleotide and a series of iron-sulfur (Fe/S) clusters. As a by-product of the reaction, the reduced flavin generates reactive oxygen species (ROS). It was suggested that the ROS generated by the respiratory chain in general could damage the Fe/S clusters of the complex. Here, we show that the binuclear Fe/S cluster N1b is specifically damaged by H2O2, however, only at high concentrations. But under the same conditions, the activity of the complex is hardly affected, since N1b can be easily bypassed during electron transfer.

Energy-converting NADH:ubiquinone oxidoreductase, respiratory complex I, plays an important role in cellular bioenergetics by coupling NADH oxidation and ubiquinone (Q) reduction with the translocation of protons across the membrane [1][2][3][4][5][6] . It consists of a peripheral arm catalyzing electron transfer and a membrane arm responsible for proton translocation. The two arms are arranged nearly perpendicular to each other resulting in an L-shaped structure of the complex. Mitochondrial complex I consists of 45 subunits including 14 core subunits that are found in all species containing an energy-converting NADH:Q oxidoreductase 7,8 . The three-dimensional structure of the core subunits of complex I is conserved from bacteria to mammals 9,10 . The bacterial complex from Escherichia coli is composed of 13 different subunits that are named NuoA to NuoN, with two of them being fused to the single subunit NuoCD 11 . They are encoded by the nuo-genes and add up to a molecular mass of approximately 530 kDa 12 . NADH is oxidized at the tip of the peripheral arm by hydride transfer to the primary electron acceptor flavin mononucleotide (FMN) 13 . From here, electrons are transferred over a distance of approximately 100 Å via a series of seven iron-sulfur (Fe/S) clusters towards the membrane, where Q is reduced and protonated in a specific binding cavity that is made up of subunits of the peripheral and the membrane arm [1][2][3][4][5][6] . A Q species is thought to move from a high-energy to a low-energy binding site inside the cavity, causing electrostatic and conformational changes that drive proton translocation in the membrane arm 2, [14][15][16] . The membrane arm contains four putative proton pathways that are connected to each other and to the Q cavity by a central axis of charged residues. It was proposed that the movement of the Q species in its cavity induces the propagation of an 'electric' wave that moves forth and back through the membrane arm triggering proton translocation 16 . Alternatively, it has been suggested that the binding of quinone leads to a transition from an 'open' to a 'closed' state 10 . Quinone reduction results in a re-distribution of protons in the membrane arm, which in turn leads to a proton release to the cytoplasm exclusively in NuoL 10 .
The ROS-producing FMN cofactor is located in the immediate vicinity of the Fe/S clusters of respiratory complex I. It is well known that solvent exposed Fe/S clusters are prone to oxidative damage 30,31 . The structures of complex I from different organisms show that its Fe/S clusters are mostly shielded from the solvent and, thus, should be protected from degradation by ROS 7-10,32-36 . Nevertheless, it was proposed that an enhanced ROS production by complex I and the respiratory chain in general may result in damage to the Fe/S clusters of

Results
Inhibition of complex I by H 2 O 2 . Due to the activity of cellular catalases and peroxidases, the H 2 O 2 concentration in the E. coli cytoplasm is in the low nanomolar range 40,41 . However, the addition of exogenous H 2 O 2 can increase the interim intracellular concentration to the micromolar range 42 . Thus, the effect of micromolar H 2 O 2 concentrations on complex I-mediated NADH oxidation was measured initially with the protein in membranes. Cytoplasmic membranes of strain BW25113Δndh nuo:nptII_FRT/pBADnuo His were obtained by differential centrifugation. Due to the lack of the alternative NADH dehydrogenase (ndh) and the disruption of the chromosomal nuo-operon, all NADH-derived activities of membranes from this strain exclusively reflect activities of wild-type complex I encoded on the plasmid.
The NADH/ferricyanide oxidoreductase activity of complex I is catalyzed by the FMN bound to NuoF and does not involve the participation of Fe/S clusters. In addition, this activity is not coupled with proton translocation. It turned out that micromolar H 2 O 2 concentrations had no effect on this activity. Only millimolar concentrations exerted a significant effect on the NADH/ferricyanide oxidoreductase activity (Fig. 1A). Titration with up to 20 mM H 2 O 2 resulted in 55% inhibition of the activity with an apparent IC 50 of 14.4 mM.
To assay the effect of H 2 O 2 on the physiological activity of complex I including electron transfer via the Fe/S clusters to Q, its influence on the NADH oxidase activity was determined (Fig. 1B). As already observed for the NADH/ferricyanide oxidoreductase activity, H 2 O 2 inhibits the NADH oxidase activity only in the millimolar range. Titration with up to 20 mM H 2 O 2 resulted in an approximately 55% inhibition of the activity with an apparent IC 50 of 13.5 mM. The similarity of both inhibition curves suggests a non-specific effect of H 2 O 2 on complex I in the membrane.
To determine whether the inhibition is reversible, an untreated aliquot of membranes and an aliquot treated with 20 mM H 2 O 2 were centrifuged three times and each was re-suspended in buffer A without H 2 O 2 . The NADH/ferricyanide and NADH oxidase activity of the treated sample was 50 ± 5% of that of the untreated in both cases. This suggests that the inhibition by H 2 O 2 is irreversible.
To specifically investigate the effect of H 2 O 2 on complex I, the protein was isolated in the presence of the detergent LMNG (see Supplementary Fig. S1) and the inhibition of the NADH:decyl-Q oxidoreductase activity of the preparation by H 2 O 2 was measured. In the range up of to 1.25 mM H 2 O 2 no effect on complex I activity was detectable (Fig. 1C). An addition of 20 mM H 2 O 2 resulted in 65% inhibition of the activity. In summary, our experiments demonstrate that at concentrations observed under physiological conditions, H 2 O 2 has no effect on the activity of complex I.

Oxidation of complex I Fe/S clusters by H 2 O 2 .
To determine whether H 2 O 2 is nevertheless capable of damaging the Fe/S clusters of the complex, a preparation was split into aliquots and samples were either treated with buffer or with an equal volume H 2 O 2 at various concentrations. The samples were reduced by a 2000 fold molar excess of NADH, incubated with H 2 O 2 for one minute at ambient temperature and then frozen in a refrigerant solution at 150 K. EPR spectra were recorded at 40 K and 2 mW microwave power to detect the two binuclear Fe/S clusters of complex I, N1a and N1b. In addition, spectra were recorded at 13 K and 5 mW to detect the tetranuclear clusters N2, N3, and N4. The other Fe/S clusters of the complex are not detectable by EPR 43 . The sample supplied with only buffer was used as reference. Samples that were first treated with H 2 O 2 and then reduced by NADH resulted in similar EPR spectra. Incubation of the samples with H 2 O 2 for 30 min either before or after reduction by NADH did not result in spectral changes.
EPR spectra of the samples that were incubated with up to 100 µM H 2 O 2 did not show any significant spectral change (Fig. 2b,e). However, the sample treated with 1 mM H 2 O 2 clearly showed a drastic and specific loss of cluster N1b (Fig. 2c,f). The difference of the spectrum of the untreated sample obtained at 40 K minus that of the sample treated with 1 mM H 2 O 2 clearly shows the signals of cluster N1b (see Supplementary Fig. S2). Thus, the proportion of N1a has not changed, as its signal would otherwise also be detectable in the difference spectrum. However, a complete oxidation of N1b cannot be deduced from the difference spectrum. The loss of cluster N1b is also seen in the spectra recorded at 13 K and 5 mW microwave power (Fig. 2c,f). Here, a residual amount N1b is still detectable in the spectra recorded at 13 K (Fig. 2f)  Solvent channels to cluster N1b. The structure of the complex from various species shows that the Fe/S centers are not solvent exposed. To find out why N1b can be attacked by H 2 O 2 nonetheless, we took a closer look at the cryo-EM structure of the peripheral arm of the E. coli complex that contains all Fe/S clusters 44 . N1b is coordinated by four cysteine residues of a typical [2Fe-2S] ferredoxin fold at the N-terminal part of NuoG (Fig. 3).   (Fig. 3). We used the program CAVER to identify possible solvent channels that might lead from the protein surface to the cluster. A channel with a diameter of 2.28 Å leads from the solvent to the cluster (Fig. 3A). The channel is flanked by residues Gly45 G , Arg46 G and Met67 G (the superscript refers to the name of the subunit of E. coli complex I). Mitochondrial complex I contains several accessory subunits, whose number depends on the particular organism. However, none of these accessory subunits further shields cluster N1b towards the solvent. Exemplarily, the environment of cluster N1b of the complex I from ovine mitochondria 36 is shown in Fig. 3B. In mitochondrial complex I, N1b is positioned about 8.2 Å beneath the protein surface. Here, the channel has a slightly smaller diameter of 2.26 Å and extends over a longer distance in a U-shaped fashion (Fig. 3B). This is due to the presence of Leu225 from subunit NDUFV1, the homologue of E. coli NuoF, and Arg53 from NDUFS1, the homologue of E. coli NuoG, that both block the direct path to the cluster. In ovine complex I, the path is further gated by residues Gly50, Arg53, Ala67 and Ala70 from NDUFS1 and Tyr118 from subunit NDUFS4, an accessory subunit that is a structural homologue of an extra-domain of NuoG 44 . The solvent channels are sufficiently large in both organisms to allow the passage of H 2 O 2 to the cluster. Some of the lining atoms are hydrophobic and hamper rapid passage, yet without blocking it. Thus, an addition of H 2 O 2 would presumably also lead to a damage of N1b in mitochondrial complex I.

Discussion
It has been previously reported that H 2 O 2 has no effect on E. coli complex I activity 42 . However, no data were shown and it was only mentioned that 5 mM H 2 O 2 do not diminish its activity. From this data it was concluded that H 2 O 2 does not oxidize the Fe/S clusters of complex I 42 . Here, we show that incubation of E. coli membranes with 5 mM H 2 O 2 leads to a small but significant inhibition of NADH oxidase activity, when complex I is overproduced. Half-maximal inhibition is achieved at 13.5 mM H 2 O 2 (Fig. 1B). The inhibition of NADH/ferricyanide oxidoreductase activity showed a similar activity course with an IC 50 of 14.4 mM H 2 O 2 (Fig. 1A). However, incubation of free FMN and NADH with 20 mM H 2 O 2 does not lead to their chemical modification (see Supplementary Fig. S4). From this, we propose that the decrease in NADH oxidase and NADH/ferricyanide oxidoreductase activities at high H 2 O 2 concentrations (Fig. 1A,B) is most likely due to unspecific protein oxidation 45 . However, in contrast to the assumption that H 2 O 2 has no effect on the Fe/S cluster of the complex 42 , we show experimentally by EPR spectroscopy that the addition of 1 mM H 2 O 2 leads to the oxidative degradation of the binuclear cluster N1b on subunit NuoG (Fig. 2). Calculations of the intramolecular electron transfer rates revealed that electron transfer from cluster N3 to N4 via cluster N1b can easily be bypassed by direct electron transfer from cluster N3 to N4, both located on NuoG (Fig. 4) 46,47 . In this case, the reduced flavin will transfer its electrons sequentially to cluster N3 as in the non-treated complex. Here, the electrons will have a sequential transient stop before they appear at clusters N4 and N5 47 . The edge-to-edge distance between N3 and N4 is 14.9 Å, which theoretically leads to an overall 10 times diminished intramolecular electron transfer rate 46,47 . This will not change the overall electron transfer rate from NADH to Q as the Q reduction and release is much slower www.nature.com/scientificreports/ than the intra-molecular electron transfer along the Fe/S clusters 43 . Recently, we generated a complex I variant that lacked cluster N1b by deleting the E. coli iron-sulfur cluster carrier protein BolA 48 . The lack of cluster N1b led to the assembly of an active complex. Importantly, the loss of N1b did not affect the NADH oxidase activity of the mutant membranes, which means that the lack of N1b did not alter the overall electron transfer rate from NADH to Q 48 . The oxidative damage of N1b by H 2 O 2 was unexpected as the Fe/S clusters of complex I are buried well within the protein. However, the short channel identified with the program CAVER paves the way for H 2 O 2 from the protein surface to cluster N1b in bacterial and mitochondrial complex I (Fig. 3). Looking at the overall structure of the complex, it appears that cluster N1a on NuoE is most exposed to the aqueous medium (Fig. 3). On the other hand, the protein environment of N1a is more hydrophobic than that of the other clusters. Indeed, CAVER identified a channel with a diameter of 1.98 Å in E. coli and 1.9 Å in ovine complex I, respectively, (Fig. 3C,D) leading from the protein surface directly to N1a. Remarkably, however, this channel is lined with apolar atoms at the respective constrictions. Most likely, this hydrophobic surface of the channel prevents H 2 O 2 from damaging cluster N1a as well.
Taken together, our data clearly show that physiological H 2 O 2 concentrations do not damage the Fe/S clusters of respiratory complex I. Furthermore, elevated concentrations specifically damage cluster N1b. However, N1b damage does not affect complex I activity, as this cluster can be bypassed during intramolecular electron transfer, thus preserving the physiological activity of the complex. Protein preparation. The complex was prepared as described 52 . In short, membrane proteins were extracted with 2% (w/v) lauryl maltose neopentyl glycol (LMNG; final concentration), the cleared extract was adjusted to 20 mM imidazole and applied to a 35 mL ProBond Ni 2+ -IDA column (Invitrogen) equilibrated in buffer A with 5 mM MgCl 2 , 10% (v/v) glycerol, 0.005% (w/v) LMNG and 20 mM imidazole at pH 6.8. Bound proteins were eluted with the same buffer containing 308 mM imidazole. Fractions containing NADH/ferricyanide oxidoreductase activity were pooled, concentrated by ultrafiltration in 100 kDa MWCO Amicon Ultra-15 centrifugal filter devices (Millipore) and polished using a Superose 6 size exclusion chromatography column (300 mL, GE Healthcare) equilibrated in buffer A with 5 mM MgCl 2 , 10% (v/v) glycerol and 0.005% (w/v) LMNG. The fractions with highest NADH/ferricyanide oxidoreductase activity were used for further studies.
Activity assays. Activity assays were performed at 30 °C. The NADH oxidase activity of cytoplasmic membranes was determined with a Clarke-type oxygen electrode (DW1; Hansatech) as described 51 . The electrode was calibrated by adding a few grains of sodium dithionite to air saturated buffer 51 . The NADH/ferricyanide oxidoreductase activity was determined as decrease of the ferricyanide absorbance at 410 nm with a diodearray spectrometer (QS cuvette, d = 1 cm, Hellma; TIDAS II, J&M Aalen) using a ε of 1 mM −1 cm −153 . The assay was performed in buffer A containing 1 mM ferricyanide and 0.2 mM NADH. The reaction was started by the addition of the protein and the rate of the enzymatic reaction was corrected by the value of the non-enzymatic reaction. The NADH:decyl-Q oxidoreductase activity was measured as decrease of the NADH concentration at 340 nm using an ε of 6.3 mM −1 cm −1 (QS cuvette, d = 1 cm, Hellma; TIDAS II, J&M Aalen). Purified complex I was mixed in a 1:1 (w/w) ratio with E. coli polar lipids (10 mg mL −1 ; Avanti) and incubated on ice for 30 min.
The assay contained 60 µM decyl-Q, 2 µg complex I and a tenfold molar excess (5 µg) E. coli cytochrome bo 3 oxidase in buffer A with 5 mM MgCl 2 , 10% (v/v) glycerol and 0.005% (w/v) LMNG. The reaction was started by an addition of 150 µM NADH 46 . Different concentrations of H 2 O 2 (30%, v/v; Chemsolute) were added to the assays just before the start of the reaction.
To determine the reversibility of H 2 O 2 inhibition, membranes were incubated for 5 min with 20 mM H 2 O 2 . Aliquots of treated and non-treated membranes were centrifuged (178,000g, 4 °C, 60 min, rotor 60Ti, Sorvall wX + ultra centrifuge, Thermo Scientific) and re-suspended in the tenfold volume buffer A with 5 mM MgCl 2 The edge-to-edge distances between the clusters in Å are indicated on the respective arrows. The lack of N1b can be bypassed by direct electron transfer from N3 to N4 (red; nomenclature according to 59  www.nature.com/scientificreports/ and 0.1 mM PMSF. This procedure was repeated two times and the NADH/ferricyanide and NADH oxidase activity of both samples were determined. EPR spectroscopy. EPR measurements were conducted with an EMX 6/1 spectrometer (Bruker) operating at X-band. The sample temperature was controlled with an ESR-9 helium flow cryostat (Oxford Instruments). Spectra were recorded at 40 K and 2 mW microwave power and at 13 K and 5 mW microwave power from 300 to 380 mT. Other EPR conditions were: microwave frequency, 9.360 GHz; modulation amplitude, 0.6 mT; time constant, 0.164 s; scan rate, 17.9 mT min −1 . 300 µL complex I (2.5-3.5 mg mL −1 ) in buffer A were reduced with a 2000 fold molar excess NADH (10-14 mM) and shock frozen at 150 K in 2-methylbutane/methylcyclohexane (1:5; v:v).
To determine whether H 2 O 2 oxidizes or damages cluster N1b, complex I was incubated with 1 mM H 2 O 2 for 5 min. The excess H 2 O 2 was removed by concentrating the sample by ultrafiltration (Amicon Ultra-15, MWCO: 100 kDa, Millipore; 3800 g, 4 °C, rotor A-4-44, centrifuge 5804R, Eppendorf) and subsequent tenfold dilution in buffer A with 5 mM MgCl 2 , 10% (v/v) glycerol and 0.005% (w/v) LMNG. This procedure was repeated two times. An EPR spectrum of the concentrated sample reduced by a 2000 fold molar excess NADH was recorded. Other analytical methods. Protein concentration was determined according to the biuret method using BSA as a standard 56 . The concentration of purified complex I was determined by UV/vis-spectroscopy (TIDAS II, J&M Aalen) using an ε of 781 mM −1 cm −1 as derived from the amino acid sequence 57 . SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was performed with a 10% separating gel and a 3.9% stacking gel 58 . A possible oxidation of FMN and NADH by H 2 O 2 was assayed by LC-MS analysis. 1 mM FMN and 1 mM NADH (both from Sigma Aldrich) in buffer A were incubated with 20 mM H 2 O 2 for 30 min at ambient temperature and then subjected to HPLC (ProntoSIL 120-3-C18; AQ plus; 1 mL, 150 × 3.0 mm) at a flow rate of 0.5 mL min −1 in 10% triethylammonium acetate (100 mM) and 90% water. After 1 min, a gradient from 10 to 90% acetonitrile was applied over 17 min. Eluting samples were directly analyzed by API-MS (Dionex MSQ Plus).

Data availability
The data supporting the findings of this article are available from the corresponding author upon reasonable request.